The characterisatio n of intestinal lactic acid bacteria using bacteriological, biochemical and molecular approaches
Date
2011-07-22
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Abstract
Piimhappebakterid (lactic acid bacteria, LAB), sealhulgas Lactobacillus spp. ja Bifidobacterium spp., kuuluvad inimese kasuliku mikrobioota hulka, säilitades seedekulgla mikroobikoosluste tasakaalu. Samal ajal kutsuvad nad oma rakkude ja ainevahetusproduktidega organismis esile üldefekte, mis võivad mõjustada inimese tervist.
Seedekulgla piimhappebakterite liigilist jaotumist ja metaboliite pole erinevatesse eagruppidesse kuuluvatel isikutel, erinevates geograafilistes piirkondades elavatel lastel ning allergilistel ja mitteallergilistel lastel uuritud. Bakterite metaboolset aktiivsust hinnatakse tavaliselt bakterioloogiliste ja biokeemiliste metoodikate abil. Seevastu molekulaarsete uurimismeetoditega saadakse täpsemaid tulemusi. Senini pole aga rakendatud kombineeritud lähenemist, et mõista mikroobide mõju inimese ainevahetusele. Uurimistöö ülesandeks oli iseloomustada bakterioloogiliste, biokeemiliste ja molekulaarsete meetodite abil soolestiku piimhappebakterite liigilist koostist ja metaboolset toimet erineva vanuse, elukoha ja tervisliku seisundiga isikutel.
Uurimisalusteks olid 79 Eesti ja 65 Rootsi 1–2 aastast last, 40 last vanuses 5aastat (20 allergilist ja 20 mitteallergilist), 24 tervet täiskasvanut ja 37 eakat isikut. Kokku uuriti 133 laktobatsilli tüve: 35 juhuslikult valitud seedetrakti tüve , 76 Eesti ja Rootsi laste seedetraktist isoleeritud tüve, 21 referentstüve ja üht L. plantarum Inducia DSM 21379 tüve. Isoleeritud laktobatsillide tüved samastati liigi tasemel feno- ja genotüübiliste meetoditega (API 50CHL ja ITS-PCR). Laktobatsillide metaboliite määrati gaaskromatograafiaga: orgaanilisi rasvhappeid (äädik-, piim-, ja merevaikhape) ja etanooli erinevates keskkonnatingimustes ning polü-ja biogeenseid amiine erinevate aminohapete keskkonnas, inimese uriinis ja probiootilises juustus. Inimeste rooja analüüsides määrati laktobatsillide olemasolu PCR meetodiga ja hulk FISH ning real-time PCR meetoditega. Allergiliste ja mitteallergiliste laste bifidobakterite liikide määramiseks kasutati liigi-spetsiifilist PCR-DGGE meetodit koos järgneva kloneerimise ja sekveneerimisega. L. plantarum Inducia DSM 21379 tüve iseloomustati fenotüübilise laktobatsillide API 50CHL samastamiskitiga, molekulaarselt ITS-PCR ja 16S rRNA sekveneerimise ning metaboliite gaaskromatograafiaga.
Täiskasvanute ja eakate kehamassi indeks arvutati kehakaalu (kg) ja pikkuse (cm) ruudu suhte alusel. Vereplasma glükoosisisaldus (mmol/L) määrati laboratoorsete standardmeetoditega.
Selgus, et erinevates biotehnoloogilistes rakendustes kasutatavate laktobatsillide omaduste kirjeldamiseks sobivad fenotüübilised meetodid, mis põhinevad bakterite ainevahetusel, nagu biokeemilise profiili kindlakstegemine API 50CHL-ga ja metaboliitide määramine gaaskromatograafiaga. Laktobatsillide liikide, eelkõige Lactobacillus acidophilus gruppi kuuluvate bakterite määramiseks on molekulaarsed meetodid nagu ITS-PCR ja 16S rRNA sekveneerimine võrreldes API 50CHLga selgelt eelistatavamad. Bifidobacterium spp. hulga määramiseks imikutel on FISH meetod märkimisväärselt kõrgema tundlikkusega kui bakterioloogiline meetod. Kuid Lactobacillus spp. hulga määramisel nii lastel kui täiskasvanutel ühtib bakterioloogilise ja FISH meetodi kõrge tundlikkus. Erinevates vanuserühmades on laktobatsillide hulgad FISH ja real-time PCR meetoditega määramisel kõrgemad võrreldes bakterioloogiliste meetoditega.
Erinevate inimeste soole indigeensete laktobatsillide hulkade ja liikide mitmekesisus on seotud erinevate geograafiliste piirkondadega ja inimese vanusega. Bakterioloogiliselt määratud Lactobacillus spp. kõrgemad hulgad seedekulglas seostuvad täiskasvanutel ja eakatel isikutel kõrgema kehamassi indeksi ja vere glükoosi sisaldusega, kusjuures erinevad laktobatsillide liigid seostuvad erinevalt glükoosi ainevahetusega.
Molekulaarsete metoodikatega kinnitusid 5 aastastel lastel eelnevate bakterioloogiliste uuringute tulemused soole bifidobakterite levimuse ja allergia seoste kohta allergilistel ja mitteallergilistel imikutel. Allergilistel lastel prevaleerib seedekulgla bifidobakterite perekonnas B. adolescentis, samas kui mitte-allergilistel lastel on sagedasem B. catenulatum/pseudocatenulatum.
Probiootikumina patenteeritud tüvi Lactobacillus plantarum Inducia DSM 21379 samastati fenotüpeerimis- (API 50CHL) ja genotüpeerimismeetoditega (ITS-PCR, PFGE), millele lisandus 16S rRNA sekveneerimine. Need uurimismeetodid on heaks võimaluseks laktobatsillide samastamisel ja tüpiseerimisel bioloogilisest materjalist ja probiootilistest toodetest. Putrestsiini märkimisväärne hulk söötmes ja Inducia juustus on vastavuses laktobatsilli tüve Inducia poolse putrestsiini metabolismiga tarbija organismis.
Saadud tulemused ja kogutud laktobatsillide tüved pakuvad edaspidiseks võimalusi uute probiootiliste toodete väljatöötamiseks.
Lactic acid bacteria (LAB), included Lactobacillus spp. and Bifidobacterium spp., are considered to be members of beneficial microbiota that is important in maintaining a healthy, balanced microbial community in the GI-tract. LAB is proposed to induce systemic effects that may improve host’s health by their cells and metabolites. The species composition of LAB and metabolites of lower parts of GI tract are still not well elaborated in different age groups and some environmental bound diseases like allergy. Usually, metabolic impact has been assessed by phenotypic and biochemical analysis. However, molecular analysis provides more precise data. There are still a few data on the combined approach for understanding the impact of microbes on host metabolism. The aim of present thesis was to apply different methods for estimation of phenotypic and genetic properties of LAB intestinal isolates for characterization of the intestinal LAB microbiota of humans at different age, geographical location and health status. Altogether 79 Estonian and 65 Swedish infants, 40 children of 5-years age (20 allergic, 20 non-allergic), 24 healthy adults and 37 elderly persons were recruited in this study. The number of Lactobacillus sp. strains investigated was 133, incl. 35 randomly collected intestinal strains, 76 intestinal strains of Estonian and Swedish children 1-2yrs , 21 reference strains and 1 strain L. plantarum Inducia DSM 21379. Lactobacilli isolates have been typed using API CHL50 kit and ITS-PCR with species-specific primers. LAB metabolites were assessed by gas chromatography (GC): SCFA (acetic, lactic, succinic acids) and ethanol in MRS medium in different environments; poly- and biogenic amines in media with different precursor amino acids (arginine; glutamine; lysine; ornitine; histidine); in human urine samples and in the probiotic cheese.The counts of LAB in human fecal samples were detected by bacteriological (specific media) and molecular (FISH, real-time PCR) methods. The Bifidobacterium genus-specific PCR in combination with DGGE, cloning and sequencing was used to detect bifidobacterial communities in allergic and non-allergic children. L. plantarum Inducia DSM 21379 has been characterized by using ITS-PCR, PFGE, 16S rRNA sequencing and the metabolites by GC methods. Body mass index (BMI) in adults and elderly was calculated as weight (kg)/height square meters. Plasma glucose (mmol/L) was determined by standard laboratory methods. It was assessed that the phenotypic methods used including culture on artifical media with subsequent biochemical profiling by API 50CHL and GC estimation of organic acids and ethanol are appropriate for estimation of the comparative Lactobacillus species composition for biotechnological applications. The molecular methods such as ITS-PCR, controlled by 16S rRNA sequencing are clearly superior to API 50CHL in species identification, particularly for the Lactobacillus acidophilus group. For quantitative estimation of Bifidobacterium spp. of infants the FISH method has remarkably higher sensitivity in comparison with bacteriological methods. At the same time, for Lactobacillus spp. the concordance between bacteriology and FISH methods is high both in infants and adults. In comparison with bacteriological data, the higher counts of lactobacilli can be detected in different age groups using FISH and RT-PCR. The composition of intestinal indigenous lactobacilli is characterised with large individual variety both in counts, number and species composition depending on the geographical location and age. The faecal microbiota of elderly people in comparison with adults express higher total counts of lactobacilli. Both in adults and the elderly the higher counts of cultivable intestinal Lactobacillus spp. can be associated with higher BMI and blood glucose content while particular species. are differentially associated with glucose metabolism. The previously bacteriologically assessed association between the shifts of prevalence of culturable bifidobacteria in infants and the development of allergy was confirmed with molecular methods in children at 5 years of age. Particularly, in allergic children, B. adolescentis is prevalent among intestinal Bifidobacterium spp., whereas in non-allergic children B. catenulatum/pseudocatenulatum is prevalent. Elaborated into probiotics and patented, the L. plantarum Inducia DSM 21379 strain was identified using phenotypic (API 50CHL) and genotypic (ITSPCR, PFGE) methods followed by 16S rRNA sequencing. These methods serve as useful tools for strain identification and typing of lactobacilli from biological material and probiotic products. The concordance between the high proportions of putrescine amounts produced by strain L. plantarum Inducia in decarboxylation media, Inducia cheese and urine of its consumer was detected. The obtained knowledge and collected large pool of well characterized Lactobacillus sp. strains can open new possibilities to elaborate novel probiotics in future.
Lactic acid bacteria (LAB), included Lactobacillus spp. and Bifidobacterium spp., are considered to be members of beneficial microbiota that is important in maintaining a healthy, balanced microbial community in the GI-tract. LAB is proposed to induce systemic effects that may improve host’s health by their cells and metabolites. The species composition of LAB and metabolites of lower parts of GI tract are still not well elaborated in different age groups and some environmental bound diseases like allergy. Usually, metabolic impact has been assessed by phenotypic and biochemical analysis. However, molecular analysis provides more precise data. There are still a few data on the combined approach for understanding the impact of microbes on host metabolism. The aim of present thesis was to apply different methods for estimation of phenotypic and genetic properties of LAB intestinal isolates for characterization of the intestinal LAB microbiota of humans at different age, geographical location and health status. Altogether 79 Estonian and 65 Swedish infants, 40 children of 5-years age (20 allergic, 20 non-allergic), 24 healthy adults and 37 elderly persons were recruited in this study. The number of Lactobacillus sp. strains investigated was 133, incl. 35 randomly collected intestinal strains, 76 intestinal strains of Estonian and Swedish children 1-2yrs , 21 reference strains and 1 strain L. plantarum Inducia DSM 21379. Lactobacilli isolates have been typed using API CHL50 kit and ITS-PCR with species-specific primers. LAB metabolites were assessed by gas chromatography (GC): SCFA (acetic, lactic, succinic acids) and ethanol in MRS medium in different environments; poly- and biogenic amines in media with different precursor amino acids (arginine; glutamine; lysine; ornitine; histidine); in human urine samples and in the probiotic cheese.The counts of LAB in human fecal samples were detected by bacteriological (specific media) and molecular (FISH, real-time PCR) methods. The Bifidobacterium genus-specific PCR in combination with DGGE, cloning and sequencing was used to detect bifidobacterial communities in allergic and non-allergic children. L. plantarum Inducia DSM 21379 has been characterized by using ITS-PCR, PFGE, 16S rRNA sequencing and the metabolites by GC methods. Body mass index (BMI) in adults and elderly was calculated as weight (kg)/height square meters. Plasma glucose (mmol/L) was determined by standard laboratory methods. It was assessed that the phenotypic methods used including culture on artifical media with subsequent biochemical profiling by API 50CHL and GC estimation of organic acids and ethanol are appropriate for estimation of the comparative Lactobacillus species composition for biotechnological applications. The molecular methods such as ITS-PCR, controlled by 16S rRNA sequencing are clearly superior to API 50CHL in species identification, particularly for the Lactobacillus acidophilus group. For quantitative estimation of Bifidobacterium spp. of infants the FISH method has remarkably higher sensitivity in comparison with bacteriological methods. At the same time, for Lactobacillus spp. the concordance between bacteriology and FISH methods is high both in infants and adults. In comparison with bacteriological data, the higher counts of lactobacilli can be detected in different age groups using FISH and RT-PCR. The composition of intestinal indigenous lactobacilli is characterised with large individual variety both in counts, number and species composition depending on the geographical location and age. The faecal microbiota of elderly people in comparison with adults express higher total counts of lactobacilli. Both in adults and the elderly the higher counts of cultivable intestinal Lactobacillus spp. can be associated with higher BMI and blood glucose content while particular species. are differentially associated with glucose metabolism. The previously bacteriologically assessed association between the shifts of prevalence of culturable bifidobacteria in infants and the development of allergy was confirmed with molecular methods in children at 5 years of age. Particularly, in allergic children, B. adolescentis is prevalent among intestinal Bifidobacterium spp., whereas in non-allergic children B. catenulatum/pseudocatenulatum is prevalent. Elaborated into probiotics and patented, the L. plantarum Inducia DSM 21379 strain was identified using phenotypic (API 50CHL) and genotypic (ITSPCR, PFGE) methods followed by 16S rRNA sequencing. These methods serve as useful tools for strain identification and typing of lactobacilli from biological material and probiotic products. The concordance between the high proportions of putrescine amounts produced by strain L. plantarum Inducia in decarboxylation media, Inducia cheese and urine of its consumer was detected. The obtained knowledge and collected large pool of well characterized Lactobacillus sp. strains can open new possibilities to elaborate novel probiotics in future.
Description
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Keywords
seedekulgla mikrofloora, piimhappebakterid, liigiline koosseis, ainevahetus, gastrointestinal microflora, lactic acid bacteria, species composition, metabolism